PROJECT SUMMARY/ABSTRACT Eukaryotic cells maintain tight control over DNA replication to guarantee that each daughter cell receives one and only one copy of the genetic material. Initiation of DNA replication more than once per cell cycle, or rereplication, results in increased gene amplification and aneuploidy, which are critical parameters that contribute to tumorigenesis and tumor progression. Genome-wide analysis of copy number variation in hematopoietic and solid cancers has identified chromosomal regions with higher frequencies of gain or amplification, which often contain pro- survival and/or oncogenes. Therefore, understanding how rereplication and amplification of specific regions emerge and potentiate cancer development and drug resistance will impact our understanding of hard to treat malignancies. The work in this proposal challenges the dogmatic interpretation of DNA replication as a once-per-cell cycle event. My research program is designed to understand how prevalent rereplication is, identify the molecular determinants that specify regions for rereplication, and uncover methods to intervene to block unwanted rereplication. The work in this proposal will rigorously test how epigenetics regulates DNA rereplication. We will apply our new technology, RerepSeq, for the enrichment and unbiased identification of regions that undergo DNA rereplication. Through developing the network of rereplicated sites we can understand what pathways and features contribute to their origins. Our initial work will focus on two epigenetically driven models of rereplication. We will identify the specific network of epigenetic regulatory factors that drives rereplication at these loci and determine how we can prevent the rereplication from happening. In five years, the work from this proposal will have established how two different epigenetic pathways, H3K27me3 and DNA methylation, regulate rereplication. This work will have uncovered fundamental properties of rereplicated regions including size, epigenetic state and functional relevance to cell biology. These two initial models will establish the procedures, reagents and tools to investigate other epigenetically regulated regions of rereplication identified through our unbiased sequencing approach, which will be the basis of future work in the laboratory. Our goal is to uncover the entire epigenetic addressing system that directs rereplication. By understanding this network, we can exploit it to prevent rereplication of drug resistance or oncogenes to facilitate better treatment.